Process for the production of antibiotic w847-a from related antibiotics

ABSTRACT

Antibiotic W847 Complex or components thereof are converted into one discrete substance, Antibiotic W847-A, by controlled alkaline hydrolysis.

United States Patent Reimann 1 PROCESS FOR THE PRODUCTION OF ANTIBIOTICW847-A FROM RELATED ANTIBIOTICS [72] Inventor: Hans Reimann, Wayne, NJ.

[73] Assignee: Schering. Corporation, Bloomfield,

[22] Filed: May 6, 1968 [21] Appl. No.: 726,738

[52] US. Cl ..424/120, 424/121 [51] Int. Cl. ..A6lk 21/00 [58] Field ofSearch ..424/121, 122, 120

[451 Oct. 10,1972

Primary Examiner-Albert T. Meyers Assistant Examiner-Daren M. StephensAttorneyStephen B. Coan, Raymond A. McDonald and Bruce M. Eisen [57]ABSTRACT Antibiotic W847 Complex or components thereof are convertedinto one discrete substance, Antibiotic W847-A, by controlled alkalinehydrolysis.

8 Claim, 8 Drawing Figures PATENTEBBCI \0 m2 3.697 .649

sum 1 or 3 40003000 2000 I500 -I I000 900 800 70o I l I l l l ITRANSMITTANCE (/o) m 4 o o 3 g 6 0 9 I0 I! I2 l3 l4 l5 WAVELENGTH(MICRONS) FIG. 1 |NFRA RED SPECTRUM OF ANTIBIOTIC W847- A 4000 3000 2000I500 -l I000 900 800 700 o0 1 ABSORBANCE WAVELENGTH (NHCRONS) 2 INFRAREDSPECTRUM OF ANTIBIOTIC W 847- B 4000 3000 2000 I500 -l I000 900 800 700I I l I l I I TRANSMITTANCE (/o) FIG. 3 INFRARED SPECTRUM OF ANTIBIOTICws47-c INVENTOR- HANS REIMANN PATENTEDHBT 101912 0 3.697.649

SHEET 2 OF 3 4000 3000 2000 I500 -l I000 900 800 700 I00 TRANSMITTANCE(70) 1 v n 1 I 1 s 7 8 9 I0 I: I2 1 304 15 WAVELENGTH (MICRONS) FIG. 4INFRARED SPECTRUM OF ANTIBIOTI C W 847C 8:0 770 6:0 5:0 40 3'0 2'0 To 0NUCLEAR MAGNETIC RESONANCE SPECTRUM OF ANTIBIOTIC ws47- NUCLEAR MAGNETICRESONANCE SPECTRUM 0 [NVEN TOR. OF ANTIBIOT'C W847 B HANS REIMANNPATENTEUNI 10 I972 I 3,697,649

sum 3 [1f 3 ab 7'0 Neio 5'0 4'.o 310 2'0 1 0 0 NUC| EAR MAGNETICRESONANCE SPECTRUM 0F ANTIBIOTIC wa4 'efo 7:0 6:0 5T0 4T0 3'0 2'0 1:0 0NUCLEAR MAGNETIC RESONANCE SPECTRUM OF ANTIBIOTIC ws47--c HANS REI MANNPROCESS FOR THE PRODUCTION OF ANTIBIOTIC W847-A FROM RELATED ANTIBIOTICSThis invention relates to the transformation of closely relatedantibiotic substances, which may be coproduced, into one discretecrystalline antibiotic substance. More specifically, this inventionrelates to a process for converting the several fractions of AntibioticW847 Complex into one fraction, Antibiotic W847-A.

Antibiotic W847 Complex exhibits broad spectrum antibiotic activity. Thecomplex contains four major fractions or components and these aredesignated Fractions A, B, C and C As will be appreciated by thoseskilled in the art, it is highly desirable to have single antibioticfractions available for use, as opposed to a complex, since one canadminister a single fraction with greater assurance of uniformity in itspotency, effects, concentration, absorption and the like. Similarly, itis desirable to have the antibiotic available as a single purecrystalline compound to provide uniform standards of purity and dosage.

The microbiological production of Antibiotic W847 Complex and itsfractions is described in commonly assigned copending application, Ser.No. 707,100filed Feb. 21, 1968 by Marvin J. Weinstein, George M.Luedemann, Gerald H. Wagman and Joseph A. Marquez, now abandoned. By themethod disclosed therein, the several fractions are isolated by tedious,lengthy chromatographic techniques. I have now discovered that FractionsB, C and C may be directly converted into pure crystalline W847-A byhydrolysis under alkaline conditions.

Antibiotic W847-A itself is present in the Antibiotic W847 Complexproduced in the fermentation broth only in a relatively small amount,i.e. less than The major component is the W847-C Present studies haveshown that W847-A has several distinct advantages as compared to themajor component W847-C in terms of better tolerance, less subacutetoxicity, better activity against gram-negative organisms, higher peakblood levels and slightly better protcctive activity in mice.

In the preferred mode of operation, the conversion process is carriedout on the components without isolating them from the W847 fermentationbroth. It can also be effected on the isolated W847 Complex (generallyobtained by extracting the broth with a water immiscible solvent andconcentrating) or on the further isolated W847-C Complex. It is alsowithin the scope of this invention, however, to effect the conversionwith the individual fractions either isolated from the W847 Complex orsynthetically produced.

Antibiotic W847 Complex can be formed by cultivating the microorganismMicromonospora Sp. W847 in an aqueous nutrient medium under submergedaerohave been designated NRRL 3274 and NRRL 3275.

These strains are available from the collection agency upon request.

The instant invention, however, is not to be construed as limited to theconversion of A-ntibiotic W847 Complex, W847-B, W847-C complex, W847-C,or W847-C only when they are produced through utilization ofMicromonospora sp. W847 var. NRRL 3274 or var. NRRL 3275. Firstly, onemay also utilize other variants of Micromonospora sp. W847 or mutantsthereof produced from this organism by mutating agents such as, forexample, high frequency radiation including x-ray and ultra-violet,actinophages and nitrogen mustard. Secondly, other microorganisms mayproduce one or more Antibiotic W847 fractions. Thirdly, syntheticallyproduced Antibiotic W847 fractions could similarly be hydrolyzed toW847-A. In other words, the process of this invention is applicable andeffective in converting antibiotic substances W847-B, W847-C, andW847-C, and their mixtures to Antibiotic W847-A regardless of how theformer substances are themselves produced.

The starting materials of this invention can each be positivelyidentified without reference to their method of manufacture. AntibioticW847 Complex possesses an antibacterial spectrum substantially as shownin Table l with substantially no diminution in potency after contact for24 hours at 37 C with any of the following enzymes: trypsin,chymotrypsin, pepsin, a amylase and penicillinase.

Antibiotic Fractions A, B, C, and C can be positively identified bytheir chemical and physical properties as listed in Table 2; theirinfrared spectra as shown in FIGS. 1, 2, 3 and 4, respectively; theirnuclear magnetic resonance (NMR) spectra as shown in FIGS. 5, 6, 7 and 8respectively; and their antibacterial spectra as shown in Table l. Theinfrared spectra were run in mineral oil (Nujol) and the moresignificant absorption peaks are set forth in Table 3 with the followingdesignations: S=strong, M=moderate, W=weak, VS=very strong, M-S=moderateto strong, brd.=broad, shp.=sharp, shd.=shoulder and s.b.=side band. TheNMR spectra were observed on a Varian A--A spectrometer on a solution(ca 0.4 ml, ca 20 mg/ml) of the sample of each fraction in deuteratedchloroform. The spectra are recorded in parts per million (PPM) fromtetramethylsilane, the internal standard. In Table l, the susceptibilityof the test organisms to the antibiotics was determined by a tubedilution assay in yeast beef broth adjustt ilo pH 8.0 with sodiumhydroxide.

TABLE 1.-ANTIBACIERIAL SPECTRUM OF ANTIBIOTIC W847 COMPLEX AND FRACTIONSA, B, Cr C2 AND C-COMPLEX Minimal inhibitory concentration (mcgJmL)Antibiotic W847 Complex Complex Frat-tion Fraction Fraction FractionC-com- Microorganism base H01 1 C2 plex Bacillus megalherium DA 7064 0.30.6 1.2 0.3 0.6 0,3 Bacillus subtilis ATCC 6633 0. 03 0.3 0. 005 0. 050. 03 0. 005 0.005 D pg r fli 12%;?616015 g Di ococcus eumomaebiglococcus fieumoniae DA 150.... 6. 0 6.0 2. 7 2. 7 12.0 27 27Enterococcus sp. DA 800 0.3 0.75 0.6 0.6 0.3 0.08 0.3 Enteracaccus sp.DA 801.. 0.3 0.75 0. 6 0. 6 0.3 0. 6 0.3 Enlerococcus sp. DA 802. 0.31.0 0.2 0.2 0.3 0. 03 0. Snrcjna lutea ATQCQZQIUH V g 0. 0075 .00750.005 0.005 0. 0075 0,0005 0. 0007 TABLE 1. C o nti nued ANTIBACTERIALSPECTRUM OF ANTIBIO'IIC W847 COMPLEX AND FRACTIONS A. B, C C; ANDC-COMPLEX Minimal inhibitory concentration (meg/ml.) Antibiotic W847Complex Complex Fraction Fraction Fraction Fraction C-com-,Microorganism base H01 A B 1 C: plex taphylococcus aureus A'ITC 127150.3 0. 75 0.08 0.2 0.3 0, um Staphylococcus aureus ATCC 6538P 0.3 0.030.6 0.2 0.03 0.005 0.03 Staphylococcus aureus ATCC 11631 0.3 0.075 0.60.6 0.3 0. 6 0.3 Staphylococcus aureus (Gtaylu 0 3 0.3 0.6 0.6 0.3 0.60.5 DA 2001 0.03 0.3 0.6 0.2 0. 03 0.03 0.03 aureus DA 2003. 0. 03 o. 03o. c 0.6 0. 015 0.6 0.3 aure'us DA 2010- 0.3 0.3 0.6 0.6 0.3 0.2 0.25 aueus DA 2014- 0.3 0.3 0. 6 0.6 0.03 0.6 0. 03 aureus DA 2018- 0.3 0. 30.6 0.6 0.075 0. 1 0. 1 Staphylococcus uureus DA 2032--. (175 3 0. 6 0.6 0. 075 0. 08 0. 08 Staphylococcus aureus DA 2033. 16 16 2.7 27 32. 027 2. 7 Streptococcus facalis DA 0. 3 0.3 0. 03 0.1 0.3 0, 005 0, 075Streptococcus moaenes DA 21. 7 1. 0 5.0 5.0 0.3 0.6 0,3 Streptococcuspyooenes DA l1.. .3 0.6 0.2 0.3 0. 03 0,3 Streptococcus pyogenes DA 120. 2 0. 2 0. 03 Mycobaclerium smegmatis ATGC 10143 7 0. 3 5.0 5.0 0.30.05 0.3 Escherichia coli ATCC 10536 6.0 16 6. 0 12. 0 12. 0 12. 0 12. 0Klebaiella pneumoniae ATCC 10031 12.0 16 6. 0 24. 0 12.0 24.0 12.0Proteus ulgaris DA 121 6- 0 16 12.0 24. 0 24. 0 24. 0 24, 0 Pseudomonusaeruainosa ATCC 8689. 6. 0 12. 0 6.0 12. 0 6.0 6.0 6. 0 Salmonellaschottmuelleri DA 10 6. 0 12.0 6.0 12.0 12. 0 12, 0 12, 0

Penicillin resistant strain. Erythromycin resistant strain. Medium:Yeast beef broth (pH 8.0). Comp1ex=Mixture of components A, B, 0 and 0TABLE 2.CHEMICAL AND PHYSICAL PROPE RTIES OF ANTIBIOTIC W847 FRACTIONSFraction A Fraction B Fraction Cr Fraction C Optical rotation [11] n 1%in ethanol- 90 92 1 104 102 Melting point 255-259 C. dec. 125135 C. dec.243-246 C. dec. 146-150 C. dec. PKa. 0.1 8. 8 8. 8 8. 6 Neutralizationeq 442 400 488 492 Elemental analysis Found Calc. Found 1 Cale. 1 FoundCalc. Found Cale.

Carbon. 60. 23 60. 25 58. 90 58. 95 60. 23 59. 98 60. 54 60. 35Hydrogen- 9. 28 9. 19 8. 94 9. 03 8. 93 8. 81 8. 60 8. 89 Nitrogen-.. 3.30 3. 19 2. 88 2. 99 2. 94 2. 92 2. 87 2. 87 Oxygen (by diflerence) 27.19 27. 36 29. 18 29.03 27. 90 28. 30 27. 99 27. 89

Empirical formula CuHsoNzOn CnHuNzOm C4EHB4N2O 7 CnHscNzOn Molecularweight. 877. 10 019. 14 961. 71 075. 20

l Monohydrate.

TABLE 3.INFRARED SPECTRA OF ANTIBIOTIC W847 FRACTIONS Fraction AFraction B Fraction C1 Fraction C2 Wave- Peak Wave- Peak Wave- PeakWave- Peak length (p) strength length strength length strength lengthstrength 2. 82-2. 90 83 W-M. 3. -3. 2. Nujol. 5. 77 S. 5. 82 5. 72 5. 5.80 (instrument artifact). 5. 88 5. 83 5. 87 WM 5. 88 shd. 6. 82 6. 82 6.82 Nuioi. 6. 80 Nujol. 7. 24 7. 25 7. 25 7. 23 Nujo] 7. 36 8.05 7. 458.00 7. 82 8. 45 7. 57 8. 53 S. brd 8. 45 8. 59 7. 82 8. 93 S. 8. 92 8.94 8. 03 9. 08 shd. 9. 02 9. 30 8. l3 9. 27 M-S 9.12 9. 62 8. '9. S. 9.32 9. l1 8. 94 10. 00 M-S. brd 9. 53 10.02 9. 05 10. 38 M. 9. 63 10. 299. 56 10. 97 M. 9. 98 10. 84 9. 11. 15 M. 0. 27 11.08 10. 13 11. 52 W.

11.02 WlVL 11.18 W-M As is apparent from Table 1, Antibiotic W847-Aerrhibits a broad range of anti-microbial activity against bothgram-positive and gram-negative microorganisms. Included in the grampositive group are pathogenic microorganisms including species of thegenus Streptococcus, Staphylococcus and Diplococcus which are known tocause many disease manifestations. Various species of Staphylococcus andStreptococcus are the organisms responsible for causing bovine mastitis.These species are readily controlled and treated by means of antibioticW847-A after a relatively brief regimen of administration. AntibioticW847-A is also active against gram-negative organisms including speciesof the genus Escherichia, Salmonella, Proteus and Pseudon'lonas. Theseorganisms are responsible for many serious disease syndromes includingurinary tract infections and diarrheas. Such syndromes are quite commonin domestic animals such as cattle, horses, sheep, swine, dogs and catsand may be effectively controlled and treated by means of antibioticW847-A.

Antibiotic W847-A suspended in 0.5% aqueous carboxymethylcellulose anddispersed by ultrasonication is active by subcutaneous administration inmice against S. aureus Gray with a PD (protective dose for 50 percent ofthe population tested) of 20 mg/kg and against P. aeruginosa with a PD:of 161 mg/kg. The LD is mice by the subcutaneous route is 7,000 mg/kg.

Antibiotic W847-A may also be used to clean and sterilize laboratoryglassware, surgical instruments and the like. It may also be used incombination with soaps and detergents to clean and sanitize areas usedfor food preparation such as kitchens, dining halls and the like.

According to the process of this invention, the Antibiotic W847 startingmaterial is subjected to hydrolysis under non-acidic conditions andspecifically at a pH in the range of about 9 to about 12 until theconversion to W847-A is substantially complete.

The completeness of hydrolysis can be checked in several ways. Oneconvenient procedure employs thin layer chromatography and consists ofremoving a milliliter aliquot of the reaction mixture and, if necessary,adding enough sulfuric acid to lower the pH to within the range of about9.0-9.5. The aliquot is then extracted with 15 ml. of ethyl acetate andthe extract concentrated to dryness under reduced pressure. The residueis then dissolved in 0.2 ml. of 95% ethanol and 1.1. (20A) of thissolution is spotted on thin layer silica gel GF plates (Analtech, Inc.)using a 40% methanol-60% chloroform solvent system. A like amount of anauthentic antibiotic W847-A sample is similarly spotted. After 15-30minutes, the plates are sprayed with a mixture of concentrated sulfuricacidmethanol (1:1 v/v) and developed by heating at 105 C for severalminutes. The position and color of the two spots are compared.

The hydrolysis rate can be increased by working at elevatedtemperatures. The use of such elevated temperatures is not practical,however, when hydrolyzing the broth directly or when ammonium hydroxideis used to render the medium basic.

Materials suitable for rendering the hydrolysis medium basic includeammonium hydroxide, sodium hydroxide, potassium hydroxide, calciumhydroxide, sodium carbonate, sodium bicarbonate and the like. Again, theamount added must be such as to produce a pH within the range of about 9to about 12.

Significant decomposition results if the hydrolysis is conducted underacidic pH condition or at a pH above about 12. Hydrolysis at a pH of atleast 7 but less than about 9 is too slow to be practical. The latterpoint is evidenced by the fact that the fermentation mixture is withinthis range, and yet after the several days required for fermentation,only a minor amount of W847-A is present (less than 10%).

As noted above, the direct hydrolysis of the fermentation broth ispreferred. Among other advantages, the procedure allows the AntibioticW847-A to be removed from the broth by extraction with about onefifththe broth volume of methylene chloride or equivalent solvent. On theother hand, when the antibiotic complex W847 is first isolated from thebroth, it requires about twice the broth volume of solvent. The brothcan be filtered before or after effecting the hydrolysis process of thisinvention. Generally a filter aid such as Celite or Supercel is addedbefore filtration.

After hydrolysis of the fermentation broth according to the method ofthis invention, the filtered broth is extracted with a water-immisciblepolar organic solvent such as methylene chloride and the extract isconcentrated. Crystallization of the concentrate may be aided byaddition of a solvent such as acetone in which Antibiotic W847-A haslimited solubility.

When the alkaline hydrolysis reaction is not performed on the wholebroth, the solvent system should preferably contain a water-misciblepolar organic component in order to increase the solubility of theAntibiotic W847 components and thus increase the rate of hydrolysis.Methanol represents such a convenient component. Other such watermiscible organic solvents are ethanol, isopropanol, acetone anddimethylacetamide. Of course, the component must be one which is notitself saponified under the reaction conditions.

Although I do not wish to be limited by my theory of the chemistryunderlying the conversion process of my invention, it appears to involvesurprisingly selective saponification reactions. More specifically,Antibiotic W847 appears to contain a lactone ring and at least fouresterifiable hydroxyl groups. In Antibiotic W847-A these four hydroxylgroups are apparently not esterified. Antibiotic fractions B, C, and Cappear to be monoacetate, diacetate and monoacetatemonopropionate estersof Antibiotic W847-A. Under the selective alkaline hydrolysis conditionsof the process of this invention, the esters are apparently saponifiedto the free hydroxyl groups without causing cleavage of the lactone ringor any other degradation or rearrangement of the antibioticmacrostructure. This is surprising in that cleavage of the lactone ringunder such conditions might have been expected. Under acidic or highlybasic hydrolysis conditions, antibiotic W847 does indeed degrade.

Example 1 illustrates the preparation of Antibiotic W847 Complex byfermentation and Examples 2 and 3 illustrate means for extracting thecomplex from the fermentation broth. Such processes are disclosed in theaforesaid copending application Ser. No. 707,100now abandoned.

EXAMPLE 1 Production of Antibiotic W847 Complex Add a 0.5 ml.lyophilized culture of Micromonospora sp. W847 to a 2 liter flaskcontaining 500 ml. of the following medium which has been adjusted to pH7.5 with dilute sodium hydroxide prior to sterilization:

Bacto-Beef Extract (Difco) 3.0 gm. Bacto-Tryptose (Difco) 5.0 gm.Dextrose 1.0 gm. Starch (potato) 24.0 gm. Bacto-Yeast Extract (Difco)5.0 gm. Calcium Carbonate 2.0 gm. Tap Water 1000.0 ml.

Incubate the flask and its contents for 72 hours at 28C on a rotaryshaker (280 r.p.m.; 2 inch stroke).

Add to a fermentor 10 liters of the following sterile production mediumadjusted to pH 7.15 to 7.25 prior to sterilization:

10.0 liters Inoculate this fermentor with 500 ml. of the 72 hour seedculture prepared above. Bring the temperature of the fennentation mediumto 31 C and agitate at 500 r.p.m. while introducing an air flow throughthe medium at the rate of 0.5 liters of air per liter of broth perminute. Increase the agitation rate to 600 r.p.m. after 24 hours and to700 r.p.m. after 48 hours. Terminate the fermentation at the end of 69hours.

At the end of this period, the potency of the produced antibioticreaches a peak which remains substantially constant. Throughoutfermentation, the pH of the fermentation mixture remains substantiallyin the range of 7.2 to 8.2. The packed cell volume reaches a constantvalue of 3.5 4.5 ml. The whole broth gives a zone diameter of 25 mm whendisc tested against S. aureus orllagrgjnqsa. p a

EXAMPLE 2 Extraction of Antibiotic W847 Complex (ChromatographicTechnique) Adjust 60 liters of whole broth prepared according to theprocedure described in Example 1 to pH 9.5 with dilute aqueous sodiumhydroxide. Extract with 2 volumes of ethyl acetate for each volume ofbroth. Separate the solvent phase and concentrate under vacuum to obtainan oily residue (approximately 30.0 g.). This oily residue at i/dilution gives a zone of inhibition diameter of about 20-30 mm againstS. aureus and about 15-25 mm against P. aeruginosa. Purify the oilyresidue by column chromatography according to the following techniques:

Prepare a column using 1,000 g. of LH20 Sephadex- (Pharmacia FineChemical, Inc.) suspended in 95 percent aqueous ethanol. Transfer theoily residue to the column and elute with 95 percent ethanol at a flowrate of 200 mg/hr. Collect 50 ml. fractions. Combine fractions accordingto their antibacterial activity (determined by agar disc testing againstS. aureus ATCC 6538P). Concentrate fractions having peak activity todryness. Dissolve the solid residue in a small amount of acetone andpour into an excess volume of petroleum ether (b.p. 30-60 C). Transferthe mixture of a dry ice-acetone bath (approximately 50 C) and allow tostand 20 minutes. Allow the mixture to return to room temperature andseparate the mother liquor from the oily residue by decanting.Concentrate the mother liquor to dryness to obtain purified AntibioticW847 Complex. 1

EXAMPLE 3 Extraction of Antibiotic W847 Complex (Acid ExtractionTechnique) alkaline by the addition of 5 percent aqueous sodiumhydroxide (about pH 8.5 to 9.0) and extract twice with 250 ml. portionsof ethyl acetate. Combine the ethyl acetate extracts and concentrateunder reduced pressure to dryness.

The following three examples illustrate the transformation process ofthis invention as applied to the fermentation broth (Example 4), theisolated W847 Complex (Example 5) and the isolated C fraction (Example6). All parts are by weight unless otherwise stated.

EXAMPLE 4 Production of Antibiotic W847-A by Direct Hydrolysis ofFermentation Broth Add concentrated ammonium hydroxide to the totalfermentation broth described in Example 1 to make the mixture 1N in NHOH, pH about 10.8 (270 ml. concentrated ammonium hydroxide per gallon ofbroth). Allow to stand at room temperature for 3 to 4 days.

EXAMPLE 5 Preparation of W847-A by Hydrolysis of W847 Complex Dissolve 1part of W847 complex (obtained as described in Example 3) in 7 parts ofmethanol and add 3 parts of water containing 0.67 parts of concentratedammonium hydroxide, giving a aqueous methanol solution which is 1N in NHOH. Allow this solution to stand at room temperature for 14 days. Add15% of Darco (by weight of starting material), stir at room temperaturefor 30 minutes, filter and concentrate to about one-third volume underreduced pressure. Extract twice with one-half volumes of methylenechloride. Concentrate the combined methylene chloride extract to lowvolume and add acetone and crystallize. Collect the crystalline productby filtration and wash with ice-cold acetone. Combine the washings withmother liquor and concentrate to obtain a second crop. Dry the produceovernight at 50 in a vacuum oven, m.p. about 250 C (dec.).

,7 iKQ l EQWW 7 Preparation of W847-A by Hydrolysis of W847-C FractionA. Preparation of W847-C Fraction Dissolve 542 grams of W847 Complex(obtained as described in Example 3) in 5.4 liters of acetone and treatthe resulting solution with 81 grams of decolorizing carbon for about 30minutes at about room temperature. Remove the decolorizing carbon byfiltration and concentrate the filtrate in vacuo to approximately 2.0liters. Prepare a slurry of about liters of ice and water and withvigorous stirring add the acetone solution. Allow the temperature of theresulting suspension to rise to about 25 C with stirring and collect theproduct by filtration. Wash the solids with a small quantity of waterand dry at about 50 C in vacuo to yield about 212 grams of AntibioticW847-C Complex.

as before. Concentrate the filtered solution underreduced pressure andadd acetone to afford a crystalline product. Heat the acetone on thesteam bath for a group consisting of Antibiotic W847-B, Antibiotic WisbimLcYVfiL ih n..29y t sr which antibiotics are characterized by:

antibacterial spectra as described in Table 1; optical rotation, meltingpoint, PKa, neutralization equivalent, elemental analysis, empiricalformula, and m ec ar W i h! ss s sfihsdi i ls 2; frared spectra as shownin FIGS. 2, 3 and 4, respectively; and nuclear magnetic resonancespectra as shown in FIGS. 6, 7 and 8, respectively, in an aqueous mediumhaving a pH in the range of about 9 to about 12 until said hydrolysis issubstantially complete.

2. A method according to claim 1 wherein prior to 15 short time topermit complete solution of impurities hydrolysis the PH adjusted to therange of and optimum crystallization of the desired product, and thencool and filter the product. Concentration of the mother liquor givesadditional crops. Wash the crystalline W847-A product with cold acetoneand dry at 50 C in a vacuum oven.

The total yield of Antibiotic W847-A is 149.6 g. (60% by weight), m.p.about 250 C (dec.). W847-A may be recrystallized from acetone to giveanalytical material, m.p. 255259 C (dec.); a D 90 (ethanol); MolecularWeight Found: 868 (benzene); Titration: NE. 435, pKa 9.0; Anal. FD: C,60.23; H, 9.28; N, 3.30 (average of two determinations).

As noted above, the W847-A component can itself be used directly as anantibiotic. It is also contemplated, however, that one can formderivatives of the thus-produced W847-A by esterification, saltformation and the like to optimize desired physical and/or about 9 toabout 12 by the addition of ammonium hydroxide.

3. A method according to claim 1 wherein the hydrolysis medium is 1N inammonium hydroxide.

4. A method according to claim 1 wherein the hydrolysis reaction isconducted in solution containing a water miscible polar organic solvent.

5. A method according to claim 1 wherein after said conversion issubstantially complete Antibiotic W847-A is isolated from the hydrolysismedium.

6. A method of producing Antibiotic W847A, which antibiotic ischaracterized by:

an antibacterial spectrum as described in Table l; optical rotation,melting point, PKa, neutralization equivalent, elemental analysis,empirical formula, and molecular weight as described in Table 2; an

infrared spectrum as shown in FIG. 1' and a nuclear magnetic resonancespectrum as shown in FIG. 5;

pharmacological pmpemes Numerous other variants comprising subjectingthe Antibiotic W847 fermentawithin the spirit of this invention will beapparent to one skilled in the art.

I claim: 1. The method of producing Antibiotic W847-A, which antibioticis characterized by:

an antibacterial spectrum as described in Table 1; optical rotation,melting point, PKa, neutralization equivalent, elemental analysis,empirical formula, and molecular weight as described in Table 2; aninfrared spectrum as shown in FIG. 1; and a nuclear magnetic resonancespectrum as shown in FIG. 5, comprising hydrolyzing an antibioticselected from the tion broth, produced by cultivating the microorganismMicromonospora Sp.W847 NRRL 3274 and NRRL 3275 in an aqueous nutrientmedium under submerged aerobic conditions until substantial antibioticactivity is imparted thereto, to hydrolysis at pH in the range of about9 to about 12 until said hydrolysis is substantially complete.

7. A method according to claim 6 wherein a filter aid is added to thebroth and the broth is filtered.

8. A method according to claim 6 wherein after said hydrolysis issubstantially complete Antibiotic W847-A is isolated from the hydrolysismedium.

2. A method according to claim 1 wherein prior to hydrolysis the pH isadjusted to within the range of about 9 to about 12 by the addition ofammonium hydroxide.
 3. A method according to claim 1 wherein thehydrolysis medium is 1N in ammonium hydroxide.
 4. A method according toclaim 1 wherein the hydrolysis reaction is conducted in solutioncontaining a water miscible polar organic solvent.
 5. A method accordingto claim 1 wherein after said conversion is substantially completeAntibiotic W847-A is isolated from the hydrolysis medium.
 6. A method ofproducing Antibiotic W847-A, which antibiotic is characterized by: anantibacterial spectrum as described in Table 1; optical rotation,melting point, PKa, neutralization equivalent, elemental analysis,empirical formula, and molecular weight as described in Table 2; aninfrared spectrum as shown in FIG. 1; and a nuclear magnetic resonancespectrum as shown in FIG. 5; comprising subjecting the Antibiotic W847fermentation broth, produced by cultivating the microorganismMicromonospora Sp.W847 NRRL 3274 and NRRL 3275 in an aqueous nutrientmedium under submerged aerobic conditions until substantial antibioticactivity is imparted thereto, to hydrolysis at pH in the range of about9 to about 12 until said hydrolysis is substantially complete.
 7. Amethod according to claim 6 wherein a filter aid is added to the brothand the broth is filtered.
 8. A method according to claim 6 whereinafter said hydrolysis is substantially complete Antibiotic W847-A isisolated from the hydrolysis medium.